Device for detection of fluorescent species

ABSTRACT

A device for detection of one or several fluorescent species, said species being contained in a medium, said medium being contained in a conduit, said device comprising a means of exciting the fluorescent species by light, said medium and conduit making up a structure that is transparent to the exciting and the emitted fluorescent light, and said device comprising one or several such structures, may be improved by letting at least part of the emitted fluorescent light be guided away from the illumination zone by total internal reflection (TIR) in said structure and collected from one end of said structure.

The present invention relates to an improvement in the kind of devicesthat are being used for detection of fluorescent species.

Fluorescence detection or fluorometry is a well established and oftenused method within analytical chemistry. The main features offluorescence detection are high selectivity and very high sensitivity,and the method is often applied to detection of trace constituents insamples of various kinds. Fluorescence detectors consist, in general, ofthree main subsystems, i/ an excitation light source and associatedoptics, ii/ a sample cell, and iii/ collection optics and lightdetector. The light source generates the light that excites thefluorescent species. The most often used light sources are highintensity lamps, like, e.g., xenon lamps or lasers. The excitationoptics transports the light from the light source to the illuminationzone, where the light excites the sample. Focusing optics is most oftenused, but also fiber optics and other kinds of waveguides, for example,may be used. When a laser light source is used, the focusing optics may,in some cases, be omitted. The sample can contain one or severalfluorescent species. The sample is, in general, present in a medium,e.g., a liquid solution, which in turn is contained in some kind ofsample cell. The sample cell may, e.g., be a compartment into which thesample is first loaded, then detected while being. stationary, andfinally withdrawn. The cell may also be part of some kind of conduit,through which the sample is transported to and from the illuminationzone. The collection optics collects the emitted fluorescent light in anefficient way, and transports it to the light detector. Also for thecollection optics, focusing elements are commonly used, but also, e.g.,fiber optics may be used. The collection, as well as the excitation,optics may also comprise some kind of device, e.g., a monochromator orone or several filters, for selection or dispersion of wavelengths. Theexcitation is most often performed at one wavelength or a fewwell-defined wavelengths, or, alternatively, the excitation wavelengthmay be scanned. The detection may be performed at one or severaldiscrete wavelengths or wavelength intervals, or scanned or dispersedover a wavelength interval, or the total amount of emitted light may bedetected. Wavelength selective detection increases the versatility andselectivity of fluorometry, and is a prerequisite in applications like,e.g., four colour DNA sequencing. There are many different kinds oflight detectors, e.g., photodiodes, diode arrays, CTD:s (charge transferdevices, including CCD:s (charge coupled devices) and CID:s (chargeinjection devices)), and photomultiplier tubes.

One of the most common and most important uses of fluorometry is as adetection method in connection with analytical methods wherein thesample is contained and transported in some kind of conduit. Suchanalytical methods include, but are not limited to, CE (capillaryelectrophoresis), LC (liquid chromatography), and FIA (flow injectionanalysis). In this context, the present invention will mainly bediscussed in connection with CE, but applications to other analyticalmethods are obvious to the skilled person. CE is a well-establishedseparation method with the possibility to analyse very small amounts ofsample, and yielding a very high separation efficiency.

Fluorescence detection, and especially LIF (laser induced fluorescence),is a well-established detection technique for CE. Lasers have two mainadvantages: i/ the high intensity of the light, and ii/ the ability tofocus the laser beam to a small spot within the capillary. It isimportant that the size of the light beam at the point of excitationdoes not contribute to band broadening: the width of CE peaks mayrequire beam diameters of 100 μm or less. In the most common, andwell-established, optical set-up, the orthogonal set-up, the capillaryis illuminated with a laser, and the emitted light is collected at 90°to the direction of the laser beam. The main concerns, in order tomaximise the sensitivity, are to maximise the light collectionefficiency, and to minimise the amount of stray light reaching the lightdetector. High collection efficiency is, in general, obtained by usinghigh numerical aperture collection optics. The term stray light is usedhere to denote all kinds of unwanted, detected light. Stray light may,to some extent, be rejected through the use of spectral and/or spatialfilters. Electrophoresis capillaries are often protected by a polymercoating, e.g., polyimide, which has to be removed before fluorescencedetection can be performed. Scattering of primary laser light may occurif there are polymer or other particles left on the capillary wall, ifthe wall is scratched, or if there are heterogeneities within the wallor the medium inside the capillary. Further, light scattering occurs atevery optical interface according to Fresnel's laws of reflection. Inparticular, the cylindrical columns ordinarily used in CE pose aproblem, since they scatter light also at 90° to the direction of thelaser beam. Also, most materials scatter light by elastic (Rayleigh)Raman molecular scattering. Scattered primary light may often, but notin all cases, be efficiently rejected by spectral filtering orwavelength dispersion. Wavelength shifted secondary light may present amore severe problem. Inelastic (Stokes shifted) Raman scattering orfluorescence emission from polymer or dirt particles on the column wall,from the column wall itself, from the medium in which the sample iscontained, or from impurities in the medium or in the sample itself maynot be easily rejected by spectral filtering or wavelength dispersion.Spatial filtering may be obtained by, e.g., shallow focal depthcollection optics and apertures. The light collection is spatiallyconcentrated to the region of the medium, while light emanating fromother regions is rejected.

For high efficiency separation methods, utilizing small diameter columnsand small samples, and yielding very narrow analyte bands at thedetector, like e.g., micro-LC and, in particular, CE, it is imperativethat the detection is performed on column and that the detection volumeis as small as possible. Use of an external detection cell with diameterlarger than the column will lead to band broadening, and coupling tosuch a cell does, in general, cause dead volumes leading to further bandbroadening. The maximum allowable detection volume for, e.g., a highlyefficient CE separation on a 100 μm column may be on the order of orless than 2 nl.

One proposed device for maximising light collection efficiency andminimising stray light is the confocal fluorescence microscope [Ju, J.et al., Anal. Biochem. 1995, 231, 131-40]. A laser beam is reflected bya low-pass dichroic beam splitter, and focused by a microscope objectiveto a very small spot, on the order of 10 μm, inside the capillary. Theemitted fluorescent light is collected by the same objective, buttransmitted through the beam splitter to the detection optics. Byfocusing the collection optics tightly inside the capillary, stray lightcontributions from the capillary wall are diminished. By placing anaperture at the focal point of the collected fluorescent light, straylight may be further rejected by spatial filtering. High lightcollection efficiency is achieved by using a high numerical aperturemicroscope objective. Drawbacks of this device include the need for verystrict mechanical tolerances, very careful optical alignment, and thesensitivity to, e.g., vibrations. These drawbacks are a consequence ofthe shallow focal depth utilized. Further, for cylindrical capillaries,the problem of focusing light and light collection in the interior of abody lacking circular symmetry is encountered.

Another proposed device for optimisation of detection sensitivity is thesheath flow cell [Swerdlow, H. et al., Anal. Chem. 1991, 63, 2835-41;Chen, D. Y. et al., J. Chromatogr. 1991, 559, 237-46]. The analyte to bedetected is eluted from the capillary, and excited immediately outsidethe end of the capillary in a stream of buffer flowing through a highpurity quartz cuvette. Since the analyte is detected post-capillary,stray light contributions from the capillary wall are omitted. Further,since the quartz cuvette may be designed with flat optical surfaces, thelight scattering problem associated with curved surfaces is omitted.High light collection efficiency is achieved by using high numericalaperture collection optics. The use of this device demands very carefulcontrol of flow conditions and flow impedances in order to maintain theintegrity of the analyte stream. Further, the presence of particles,bubbles, or impurities in the sheath flow buffer may lead to largeamounts of stray light.

In order to increase the sample throughput of CE analysis, like, e.g.,for large scale DNA sequencing, it is desirable to run CE in a multitudeof capillaries simultaneously. Such multiplexed analysis brings aboutseveral additional optical and geometrical problems with regard tofluorescence detection. Most often, the multitude of capillaries arearranged side-by-side in a parallel fashion, so that the array ofcapillaries form a planar array at the detection point.

The conventional on-column orthogonal set-up may be applied to capillaryarray detection [Ueno, K. et al., Anal. Chem. 1994, 66, 1424-31;Carrilho, E. et al., Proceedings of the Society of Photo-OpticalInstrumentation Engineers 1997, 2985, 4-18]. However, problems withillumination are encountered. If the planar array of capillaries isilluminated by, e.g., a number of parallel laser beams or a line-focusedlaser beam, the exciting light may form a plane that is orthogonal tothe plane of the capillary array. With this geometry, there is noorthogonal direction left for the collection of light. A 90° anglebetween exciting and emitted light may be obtained by tilting the arrayof capillaries, but such designs lead to the generation of excessivestray light as well as problems with light collection efficiency. As analternative, the capillary array may be illuminated by one single laserbeam in the same plane as the array, which laser beam hits the differentcapillaries in a subsequent manner [Anazawa, T. et al., Anal. Chem.1996, 68, 2699-2704; Yeung, E. S. et al., U.S. Pat. No. 5,741,411,1998]. With this design, the collection optics may be placed at 90° tothe incoming beam. However, since the incoming beam will hit a multitudeof optical interfaces, a lot of stray light is generated. Additionally,since some laser power is lost at each interface, the available laserpower will rapidly drop as the laser beam travels through the multitudeof capillaries, leading to a decreased fluorescence signal. Further,since laser beams are divergent, it is not possible to keep a tightfocus over an extended distance of the beam. The result is that somecapillaries will be illuminated by a not so tightly focused beam, whichmay cause detection band broadening and loss of separation resolution ofthe electrophoretic peaks.

The principle of the confocal microscope may also be applied tocapillary array detection [Mathies, R. A. et al., U.S. Pat. No.5,274,240, 1993; Kheterpal, I. et al., Electrophoresis, 1996, 17,1852-59]. In this case, the focused laser beam has to be scanned overthe capillary array (or vice versa). Thus, it is necessary to use movingparts in the detector, which is not desirable, especially in view of thedemanded tight mechanical tolerances and the susceptibility tovibrations. Further, since the laser power is shared in time between allthe different capillaries, the duty cycle per capillary is low, whichdecreases the total light collection efficiency per capillary. Theseproblems are particularly pronounced when using large arrays ofcapillaries.

Also the sheath flow cell [Takahashi, S. et al., Anal. Chem. 1994, 66,1021-26; Dovichi, N. J. et al., U.S. Pat. No. 5,567,294, 1996; Dovichi,N. J. et al., U.S. Pat. No. 5,741,412, 1998; Takahashi, S. et al., U.S.Pat. No. 5,759,374, 1998] may be applied to capillary array detection.However, specific drawbacks are encountered. Again, the simpleorthogonal setups discussed above can not be used. One possibility is toilluminate the planar array of analyte streams orthogonally with a planeof light (e.g., a line focused laser beam), and collect the emittedlight in the same plane as the capillary array, i.e., end-on collectionwith respect to the capillaries. However, fundamental opticalconstraints limit the efficient collection of light from an extendedline of objects (i.e., the capillary ends). In order to decrease thelongest dimension of the array of capillary ends, an alternative is toarrange the capillaries in a three dimensional array, i.e., in a bundle,but then the laser beam will interact with the samples over an extendeddistance, which may lead to divergence, loss of focus, and detectionband broadening. Also, tight bundling of many capillaries may result inband broadening due to inefficient dissipation of Joule heat. Further,sheath flow detection in connection with capillary arrays put extremedemands on the sophistication, control, and tolerances of the flowsystem.

A multicapillary DNA sequencing device based on transverse illuminationand guiding of the emitted fluorescent light by total internalreflection (TIR) in the capillaries has also been proposed [Takubo, K.,JP Patent No. 10019846, 1998]. However, no optical coating on thecapillaries was proposed, so TIR conditions will not be fulfilled. Sincethe refractive index (RI) of the gel inside the capillaries in mostpractical cases will be approximately equal to the RI of the electrolytebuffer, into which the capillary ends are immersed, most of the lightwill escape radially through the circumference of the capillaries, andwill not reach the capillary ends. Also, bundling of capillaries withoutoptical coating will give rise to severe optical crosstalk between thecapillaries, again preventing most of the light from reaching the end ofthe capillary in which it was emitted. Further, bundling of capillariesall the way from the injection end to the detection point will give riseto substantial Joule heating, impairing the electrophoretic resolution.

Fiber optics may also be used to transport the exciting light andcollect the emitted light [Quesada, M. A. et al., Electrophoresis, 1996,17, 1841-51]. However, alignment of a large number of individual fibersand capillaries involves a huge amount of work. Further, the amount ofstray light may be expected to exceed that of the confocal scanner orthe sheath flow cell.

The present invention is based on the idea that a device for detectionof one or several fluorescent species, said species being contained in amedium, said medium being contained in a conduit, said device comprisinga means of exciting the fluorescent species by light, said medium andconduit making up a structure that is transparent to the exciting andthe emitted fluorescent light, and said device comprising one or severalsuch structures, may be improved by letting at least part of the emittedfluorescent light be guided away from the illumination zone by totalinternal reflection (TIR) in said structure and collected from one endof said structure.

Such a device offers simplicity and robustness with respect tomechanics, optics, and liquid handling, as well as high light collectionefficiency, low stray light, and easy adaptability to capillary arraydetection.

For light travelling in a material with refractive index n₁ and strikingthe surface of a material with refractive index n₂ at an angle αto thenormal to the surface, TIR occurs if

n₁ sin α>n₂

Thus, n₁ has to be larger than n₂. Under conditions of TIR, all of thelight is, in principle, reflected back into the first material. Forereflection at angles smaller than α, some of the light is reflected andsome is transmitted.

Thus, in one aspect, the present invention provides a devicecharacterized in that the distance between the illumination zone and thelight collection end of said structure is large enough to allow lightrays emanating from the illumination zone, which do not fulfil theconditions for TIR, to be efficiently transmitted out of the lightguiding part of said structure before reaching the light collection end.Such a device ensures that only light guided by TIR through thestructure will be collected and detected at the end of the structure. Byusing a suitable arrangement, most of the exciting, primary light andpart of the stray light can be forced not to fulfil the conditions forTIR, and to be transmitted out of the light guiding structure beforereaching the light collection end.

In another aspect, the present invention provides a device that ischaracterized in that the distance between the illumination zone and thelight collection end of said structure is at least four times, orpreferably at least eight times, or even more preferably at leastsixteen times, larger than the largest cross sectional dimension of thelight guiding part of said structure. If said distance is four timeslarger, most of the light that reaches the light collection end willhave been subject to at least one reflection event. However, since therejection of light that is not subject to TIR is more efficient uponmultiple reflection events, the values eight or sixteen are morefavourable.

Further, if the illumination takes place in the immediate vicinity ofthe light collection end, scattering and diffraction of the primaryexciting light due to edge effects may cause increased levels of straylight. Even, e.g., a well-focused laser beam has a finite extent, andsuch effects may occur close to sharp edges. The present inventionprovides for a means of avoiding such effects.

The expression “species” is use to denote any fluorescent entity, suchas molecules, ions, supra-molecular aggregates, micelles, particles, orwhole cells or parts of cells. The expression “medium” is used to denotea liquid of high or low viscosity, a semi-rigid gel, or a solidmaterial. The expression “conduit” is used to denote any elongatedentity physically containing said medium, in which entity said speciesmay be transported, such as, but not limited to, a tube, a capillary, acolumn, or a channel formed in, e.g., glass, quartz, silicon, or anorganic polymer. In one particular case, the conduit is a separationcolumn for CE or LC. Said medium may or may not be transported withinsaid conduit. The exciting light may be within the ultraviolet, visible,near-infrared or infrared range. The expression “structure” is used todenote any entity comprising and physically defining said conduit andsaid medium. The term “light guiding part of the structure” denotes thatpart of the structure that is actually guiding the light, and may referto the medium, the conduit, or the medium and the conduit. Theexpression “transparent” means that the material must be able totransmit light with low loss, i.e., not highly absorbing and not highlyscattering at the relevant wavelengths. The “light collection end of thestructure” is that end where the guided light rays leave the structureand may be collected and detected by optical means. This mode of lightcollection excludes the decoupling of light from the structure by meansof any external optical decoupler before reaching the end of thestructure. An example of such a decoupler may be an optical fiberpigtailed onto the structure. In one particular case, the lightcollection end is one end of a separation column for CE or LC. The“illumination zone” is the location where the exciting, primary lightinteracts with the structure and excites the fluorescent species.

The advantages of the invention will be better understood from thefollowing discussion of the beneficial influence of different aspectsand embodiments of the invention. Clarifying examples will mainly referto detection in connection with CE, but, as will be apparent to theskilled person, the invention is not limited to such detection.

Reference is being made to the accompanying drawings, wherein:

FIG. 1 is a schematic drawing of a light guiding structure in which theRI of the conduit is larger than that of the medium.

FIG. 2 is a schematic drawing of a light guiding structure in which theRI of the conduit is smaller than that of the medium.

FIG. 3 is a schematic drawing of spectral resolution of light collectedfrom a capillary.

FIG. 4 is a ray-tracing diagram for light emanating from differentlocations in a light guiding structure.

FIG. 5 is a schematic drawing of exciting light being focused beforereaching a capillary.

FIG. 6 is a schematic drawing of a number of capillaries arranged in theform of a planar array.

FIG. 7 is a schematic drawing of light being spatially dispersed beforereaching a planar array of capillaries.

FIG. 8 is a schematic drawing of a planar array of capillaries beingrearranged into a square array at the light collection ends.

FIG. 9 is a schematic drawing of a densely packed capillary array beingimaged onto a light detector.

FIG. 10 is a schematic drawing of a sparsely packed capillary arraybeing imaged onto a light detector.

FIG. 11 is a schematic drawing of the device used in the Examples.

FIG. 12 is a trace of the fluorescent signal recorded during the pumpingof a fluorescein solution through a capillary.

FIG. 13 shows images of the light collection end of a capillary duringpumping of water and fluorescein, respectively.

FIG. 14 is an electropherogram of a fluorescein injection.

FIG. 15 is a DNA sequence obtained with the device of FIG. 11.

In one embodiment of the invention, the refractive index (RI) of themedium containing the fluorescent species is lower than the RI of theconduit containing the medium. Further, the RI of the conduit is largerthan the RI of the material surrounding the conduit. This may beillustrated by, e.g., the very common case of an aqueous solution in afused silica capillary, the capillary being surrounded by, e.g., air ora low RI polymer. In this case, TIR will not take place at the boundarybetween the medium and the conduit, but at the boundary between theconduit and the surrounding material (and to some extent at the boundarybetween the conduit and the medium). The absorbance of the surroundingmust not be too high, and the reflecting surface to the surrounding mustnot be too scattering, since this will impair the efficiency of TIR. Thelight will be guided in both the medium and the conduit. An example ofthis embodiment is shown in FIG. 1, where fluorescent light emitted fromthe illumination zone (3) is guided through the conduit (1) and themedium (2) to the light collection end (4).

In a preferred variant of this embodiment, the conduit is made of glass,fused silica, quartz, or an organic polymer. These are very common andpractical construction materials for conduits, and do, from an opticalpoint of view, allow for a range of different media, including water andmany organic solvents, to be used.

In one aspect of this variant, the conduit has an organic polymercoating. Coating of, e.g., CE capillaries renders the conduits robustenough for most practical handling, and protects the conduits from dirtand scratches. Further, the polymer coating may provide a well-definedoptical surface of high quality. Preferably, the conduit is made offused silica and the coating is a fluoropolymer. The use of fused silicacapillaries is well established within CE and high purity fused silicais an excellent optical material. Fluoropolymers do, in general, have alow refractive index, which allows for a high light collectionefficiency.

By using a transparent polymer coating of high optical quality, it isnot necessary to remove the coating at the illumination zone. Theexciting light may simply be directed through the coating and onto theconduit. The used coating must not be highly fluorescent at the employedwavelengths. The most common coating material for CE capillaries ispolyimide. This material is fluorescent and not transparent, and has tobe removed before excitation. The removal of the coating involves anextra, complicated manufacturing step. After removal of the coating, thecapillaries are mechanically very fragile, and the surface of thecapillaries is sensitive to dirt and scratches. Small, remainingpolyimide particles, may give rise to large amounts of light scatteringand background fluorescence.

In another aspect of the present variant, the conduit does not have acoating, but is surrounded by a liquid or a gas. In this way, thecoating step may be totally omitted. In CE, the liquid, may or may notbe one of the electrolyte solutions. The conduit may be, e.g., theuncoated end of a coated capillary. By using a gas, the lowest possibleRI value of the surrounding is obtained. This allows for the most highlyefficient light collection by TIR, and for the widest range of conduitmaterials to be used.

In another embodiment of the invention, the RI of said medium is largerthan the RI of the conduit, so that TIR takes place at the boundarybetween the medium and the conduit. This boundary has to be of goodoptical quality. In addition, TIR may or may not take place at theboundary between the conduit and the material surrounding the conduit.The first case is analogous to the previously described embodiment. Inthe second case, the surrounding material should be absorbing, and/orhave a higher RI than the conduit. This embodiment allows for othercombinations of materials for the conduit and the medium to be used. Anexample of this embodiment is shown in FIG. 2, where fluorescent lightemitted from the illumination zone (3) is guided through the medium (2)to the light collection end (4).

In one variant of this embodiment, the main component of the medium iswater, and the conduit is made of an organic polymer, preferably afluoropolymer or a silicone polymer. This variant makes possible the useof simple, cheap polymer tubing. Since the conduit does not guide thelight in this case, the outer shape and dimensions of the conduit arenot of primary concern. The conduit may be, e.g., a channel in a pieceof polymer material.

In another variant of this embodiment, the main component of the mediumis an organic liquid, and the conduit is made of an inorganic material,preferably glass, fused silica, or quartz. This variant allows for alarge range of different media to be used, with the only restrictionthat the RI is higher than that of the conduit. One such conceivablecombination is dimethylsulfoxide in a fused silica capillary.

In one embodiment of the invention, the conduit has the shape of ahollow cylinder. This shape is advantageous for efficient transport oflight by TIR. In analogy with optical fibers, light can be transportedover long distances in cylindrical. light guides. The cylindrical caseis a very common one; the conduit may be, e.g., a round CE capillary, anLC column, or a piece of LC or FIA tubing. Further, it is simple inpractice to design systems with cylindrical light guides.

For this embodiment, it is straightforward to calculate the lightcollection efficiency of the device. The equation for the numericalaperture (N.A.) of optical fibers is:

N.A.=(n_(core) ² −n _(coating) ²)^(0.5)

Thus, as an example, realising that other values of N.A. may be obtainedfor other combinations of materials, for a light guide with core RIequal to 1.36 (e.g., a water based buffer or hydrogel) and coating RIequal to 1.31 (e.g., a fluoropolymer), the N.A. is 0.37, equal to a highN.A. optical fiber. Obviously, the invented device may yield an adequatelight collection efficiency. Further, such a value of N.A. is compatiblewith common collecting optics, like, e.g., condenser lenses. Collectingoptics with equal or higher N.A. have been reported in some on-column,orthogonal and confocal setups. However, light leaving a cylindricalsilica capillary through the cylindrical outer surface will divergeheavily when passing the silica/air interface, so the actual lightcollection efficiency of such systems may be significantly lower.

In a preferred variant of this embodiment, the inner diameter of thecylinder is less than or equal to 500 μm, or more preferably less thanor equal to 100 μm. This is the case for, e.g., capillary columns andcapillary tubing for micro-LC and for CE. The present invention providesa means of efficiently collecting, guiding, and detecting light even forvery narrow-bore tubing.

In one embodiment of the invention, the light that is collected from theend of said structure is spectrally resolved. Spectral resolutionenhances the versatility and the selectivity of the device. Preferably,spectral resolution is performed by means of one or several prisms,gratings, or optical filters. It may be advantageous to first collectand collimate the light leaving the light guiding structure by means offocusing optics. Primary light may be blocked by, e.g., interference orlow pass filters. An example of this embodiment is shown in FIG. 3,where light exiting a capillary (5) is collected by a lens (6) andspectrally dispersed by a prism (7) before being focused by a secondlens (6) onto the light detector (8).

In one embodiment of the invention, the light that is collected from theend of said structure is detected by an imaging light detector,preferably a CTD or a photodiode array. An imaging detector consists ofseveral detector pixels, and is able to render an image of thegeometrical distribution of light. By using such a detector, an image oflight leaving the light guiding structure at different positions andangles may be obtained. This may be advantageous in some cases, e.g.,for rejecting stray light or when using a multitude of light guidingstructures.

In one embodiment of the invention, the light that is collected from theend of said structure is spatially resolved, preferably by use of anaperture or by rejecting part of a detected image. An aperture iscommonly used within photography or microscopy to perform spatiallyresolved detection of light. An imaging detector may perform the sametask: only those pixels containing the desired information is read outor stored in memory, while the signal from other pixels is rejected. Ofcourse, spatial resolution may also be accomplished by selecting theappropriate size and position of a non-imaging detector (e.g., a singlephotodiode), but this may often prove impractical.

An example of the beneficial influence of this embodiment is shown bythe ray-tracing calculation depicted in FIG. 4. The calculation is madefor a cylindrical conduit with outer diameter 375 μm, inner diameter 100μm, RI 1.46, filled with a medium of RI 1.36 in the inner channel, andsurrounded by air. In FIG. 4a, a ray-tracing calculation for a number oflight rays emanating from the center of the structure, as is the casefor emission of fluorescent light in the inner channel, was performed.The figure, showing a cross section of the capillary, illustrates thedistribution of internally reflected light rays within the capillary atsome distance away from the illumination zone. The rays travel mainlyclose to the center of the capillary, and the light intensity isespecially high within the gel-filled inner channel. FIG. 4b shows thesame calculation for a number of rays emanating from a point close tothe outer surface of the structure, as is the case for scattering ofprimary light hitting the outer surface. The rays travel mainly close tothe circumference of the capillary. By imaging the end of the structureonto an imaging detector, and selecting only pixels covering the centerregion, the scattered light may to a large extent be rejected, while thefluorescent light is efficiently detected. Clearly, the presentinvention provides a means of efficiently separating stray light,emanating from regions outside of the medium, from fluorescent light,emanating from inside the medium, by spatial resolution, using theset-up in the example, or one of a multitude of other setups.

In one embodiment of the invention, the exciting light is light from alaser. Lasers possess the advantages of high intensity and well-definedexcitation wavelengths. Further, laser light may easily be focused downto very small dimensions, which is advantageous in connection withnarrow capillaries. For the purposes of the present invention, thehighly collimated light from lasers provide an extra advantage: theillumination geometry may easily be controlled, and the amount ofprimary light coupling to the light guide through TIR may be kept verylow.

In one embodiment of the invention, the exciting light is focused in adirection parallel to the guiding direction of the emitted fluorescentlight along said light guiding structure. Preferably, the width of theexciting beam (in the said direction) should be less than 500 μm, andmore preferably less than 200 μm. For a discrete capillary, e.g., thelight is focused in the axial direction of the capillary, which is thesame direction in which light is guided. In most cases, this directioncoincides with the transport direction of the sample in the conduit. Asmall axial excitation length and a small excitation volume areimportant in, e.g., CE, where the separation efficiency is high and theanalyte bands are very narrow. In addition, the exciting light may, ormay not, be focused in an orthogonal direction to said direction. Forone, discrete capillary, e.g., the light may be focused in twodirections by means of an ordinary, round lens. For a multitude ofcapillaries or other structures, the light may be focused in only onedirection (line-focused) by means of a cylindrical lens. An example ofthis embodiment is shown in FIG. 5, where the exciting light is focusedby a lens (9) before reaching the capillary (5). The double headed arrowshows the direction in which light is guided in the capillary.

In one embodiment of the invention, the angle between the propagationdirection of the exciting light and the guiding direction of the emittedfluorescent light along said light guiding structure is large enough,preferably orthogonal or nearly orthogonal, to prevent any non-scatteredcomponent of the exciting light to be optically coupled into the guidingdirection of the light guiding structure by total internal reflection.In order to keep the level of stray light low, the amount of primary,exciting light coupling into the light guide by means of TIR should bekept as low as possible. The obvious way to achieve this is to keep theangle a for the exciting light as low as possible, which equalsorthogonal or nearly orthogonal illumination. For a well-collimated beamand a low angle α, it is (ignoring light scattering) possible to keepthe amount of TIR coupled primary light extremely low.

In one embodiment of the invention, a multitude of said structures arearranged in the form of an array, preferably a planar or nearly planararray, at the illumination zone. This may be the case, e.g., formultiplexed CE. Since light is guided by TIR within each separatestructure, the present invention is well suited for array detection. Thepresent invention has several advantages with respect to multiplexeddetection, as will be further discussed below. An example of thisembodiment is found in FIG. 6, where a number of capillaries (5)arranged in the form of a planar array are shown from different views.

In one variant of this embodiment, the exciting light is spatiallydispersed across said array. This variant is especially advantageous inconnection with planar arrays. The direction of the exciting light isorthogonal or nearly orthogonal to the planar array. The light isgeometrically spread out across the array, preferably by means of one orseveral lenses, a beam expander, or a diffractive beam shaper. The lightmay, or may not, be focused in a direction orthogonal to the dispersiondirection, e.g., by means of a cylindrical lens. This illuminationgeometry provides a very simple and clean illumination: the excitinglight passes very few optical surfaces, resulting in a low amount ofscattering. In contrast hereto, exciting light travelling in the planeof the planar array will hit the multitude of structures in a subsequentmanner, and will pass many optical surfaces, giving rise to a largeamount of scattering. Further, even though laser beams can be tightlyfocused, laser beams are divergent. The more tightly focused the beamis, the more divergent it becomes. In order to keep the axial excitationlength or the excitation volume small, it is essential to keep the beamtightly focused, and so it is essential to keep the interaction lengthbetween the beam and the multitude of structures small. This is achievedwhen the exciting light is orthogonal or nearly orthogonal to the planararray, but not when the beam hits the multitude of structures in asubsequent manner. An example of this variant is shown in FIG. 7, wherelight from a laser (10) is spatially dispersed in two dimensions bymeans of a beam expander (11) an focused in one dimension by acylindrical lens (12) before reaching the planar array of capillaries(5). The hatched areas show the approximate extent of the laser beamfrom different views.

In another variant of this embodiment, the exciting light is scannedacross said array. Again, the direction of the exciting light may beorthogonal or nearly orthogonal to a planar array. In this way, theexciting light is shared between the different structures in time ratherthan in space. The light is preferably focused in two directions bymeans of one or several ordinary, round lenses. Either the light beammay be scanned, or the array may be scanned. One alternative is to placethe multitude of structures in a circular array, and to rotate theoptics inside this array. This variant has the same advantages as theprevious one. It is also possible to use combinations of these twovariants, e.g., a scanning system in combination with a diffractive beamsplitter.

In one embodiment of the invention, the light collection ends of saidarray, preferably planar or nearly planar, of structures aregeometrically rearranged in a way that is advantageous for the efficientcollection of light, preferably in the form of a two dimensional array.Fundamental optical constraints limit the efficient collection of lightfrom an extended planar array of, e.g., capillary ends. High numericalaperture lenses collect light efficiently from a localised region; lownumerical aperture lenses have poorer collection efficiency but a widerfield-of-view. By rearranging the ends of the structures, e.g., thecapillaries, in a more compact form, e.g., a square, rectangular, orother polyhedral array, it becomes possible to collect light from alarge number of structures with a high efficiency. As an example, adensely packed planar array of 100 CE capillaries with an outer diameterof 0.5 mm is 50 mm wide, and causes difficulties with respect to lightcollection. On the other hand, if the capillary ends are rearranged intoa densely packed square array, the largest array dimension becomes only5 mm, which makes light collection significantly easier. By using thelight guiding principle of the present invention, it is possible torearrange the geometrical set-up of the multitude of structures inbetween the illumination zone and the light collection ends. A multitudeof, e.g., fused silica capillaries, may easily be rearranged from aplanar array to a square array over a distance of a few centimeters byslightly bending the capillaries. Such slight bending does notsignificantly affect the light guiding ability of the capillaries.Arranging the multitude of structures into a two dimensional arrayalready at the illumination zone will cause problems with respect to thefocusing of laser beams over extended distances and with respect tolight scattering caused by the exciting light hitting many opticalsurfaces, as discussed above. An example of this embodiment is shown inFIG. 8, where a number of capillaries (5) are arranged in a planar arrayat the illumination zone (3), but rearranged into a square array at thelight collection ends (4).

It may be noted, that dense packing of many capillaries generally causesband broadening due to inefficient dissipation of the evolved Joule heatin CE separations. In the present case however, the capillaries aredensely packed only after the point of excitation, so Joule heating willnot impair the measured separation efficiency.

In one variant of this embodiment, said two dimensional array is denselypacked, and the collected light is spectrally resolved by means of oneor several optical filters. If a densely packed two dimensional array isimaged onto the surface of an imaging detector, the image may occupy aconsiderable continuous area on the surface, and there may not be enoughspace left on the surface for wavelength dispersion of individualstructures in one dimension by means of, e.g., a prism or a grating. Inthis case, spectral resolution may be obtained by means of one orseveral filters, e.g., one or several high or low pass filters orinterference filters. The filters may be arranged, e.g., as a train offilters [Kheterpal, I. et al., Electrophoresis, 1996, 17, 1852-59] or ona rotating filter wheel. An example of this variant is shown in FIG. 9.FIG. 9a shows a number of capillaries (5) that are densely packed. FIG.9b shows light from the capillaries being collected by a lens (6) andpassed through a filter (13) on a rotating filter wheel, before beingimaged on the detector (8). FIG. 9c shows the image of the capillaryarray on the detector at one defined point of time.

In another variant of this embodiment, said two dimensional array issparse enough to allow for the collected light to be spectrally resolvedonto the surface of an imaging detector by means of one or severalprisms or gratings. The image on the surface of an imaging detectorbecomes sparse enough, so that there is space in between the image ofindividual structures for spectral resolution in one direction. Anexample of this variant is shown in FIG. 10. FIG. 10a shows a number ofcapillaries (5) that are sparsely packed. FIG. 10b shows light from thecapillaries being collected by a lens (6) and passed through a prism (7)before being imaged on the detector (8). FIG. 10c shows the spectrallyresolved image of the capillary array on the detector. The round imagesof individual capillaries are stretched out due imaging of differentcolours on different spots on the detector.

In one embodiment of the invention, using a multitude of saidstructures, the individual structures may be identified by couplinglight by total internal reflection into the structures at some pointother than the light collection end, and collecting said light from thelight collection ends of said structures. If, e.g., a large number of CEcapillaries are bundled together and imaged at the light collection end,it may be difficult to identify which injection end belongs to aspecific imaged light collection end. However, by shining light, e.g.,from a light emitting diode, onto each capillary, on at a time, at theinjection end, letting the light be guided to the light collection endby TIR, and monitoring the image on the detector, this problem may besolved. Thus, it becomes possible to bundle a large number ofcapillaries together in a non-systematic and random way, and to identifythe individual capillaries afterwards.

In addition to what is explained above, it is obvious that the presentinvented device, as compared to the on-column orthogonal optical set-up,provides a simple and efficient means of obtaining high light collectionefficiency and of separating primary light and stray light from emittedfluorescent light, especially in connection with multiplexed detection.In comparison with the confocal microscope, the focal depth is notcritical, and so the robustness with respect to mechanical tolerances,optical alignment, and, e.g., vibrations is significantly higher. Formultiplexed detection, no moving parts are necessary for the presentinvention. In comparison with the sheath flow cell, the present devicedoes not put as high demands on the sophistication, control, andtolerances of the flow system, and so offers a significantly higherdegree of robustness. Further, the present device offers a simplesolution to the problem of illumination of a multitude of capillaries.

Internally reflected light may, in principle, be decoupled from thestructure through its circumference by means of an external opticaldecoupler, and collected before reaching the end of the structure. Sucha decoupler may be, e.g., an optical fiber pigtailed onto the structure.However, such decoupling has several drawbacks. The structure must notbe coated, or the coating has to be removed at the decoupling point. TheRI of the medium must not be higher than that of the conduit. Opticaldecoupling does not provide for any means of stray light rejection like,e.g., the sheath flow cell and the confocal microscope do. Since spatialinformation is lost on decoupling, any rejection of stray light byspatial resolution is impossible. Rather, since stray light may travelmainly close to the circumference of the structure and fluorescent lightmainly close the center, decoupling through the circumference of thestructure may lead to enrichment of stray light. Further, opticalalignment of a large number of decouplers to a multitude of structuresinvolves a huge amount of work, and the decouplers may occupy aconsiderable space, making this arrangement less well suited formultiplexed detection.

There are several applications of the invented device. A few exampleswill be given, but the applicability is not limited to these examples,and other applications will be obvious to the skilled person. The devicemay be used for the detection of species with native fluorescence aswell as species labelled with one or several fluorophores. Nativefluorescence in the UV region is exhibited, e.g., by the amino acidstryptophan and tyrosine. Labelling with two fluorophores may refer,e.g., to the use of energy transfer fluorophores, with one donating andone accepting fluorophore [Ju, J. et al., Nature Medicine 1996, 2,246-49]. The device may be used for detection in connection with anymethod involving the transportation of said species across theillumination zone within said conduit. Such methods are very commonwithin chemical and biochemical analysis. The sample may, e.g., betransported within a length of capillary tubing by means of pressure,electroosmotic flow, or migration in an electric field. The device may,e.g., be used for detection in connection with capillaryelectrophoresis, including capillary zone electrophoresis, capillary gelelectrophoresis, micellar electrokinetic capillary chromatography, andcapillary isoelectric focusing, capillary electrochromatography, liquidchromatography, or flow injection analysis. The device may be used fordetection in connection with nucleic acid analysis, e.g., in connectionwith DNA sequencing. One application of special interest is highthroughput DNA sequencing by CE in arrays of capillaries.

The method of the invention will now be illustrated by the following,non-limiting examples.

EXAMPLES

The device used in the examples is shown in FIG. 11.

In all examples, fluoropolymer coated cylindrical fused silicacapillaries (14) with i.d. 100 μm and o.d. 375 μm (TSU100375, Polymicro,Phoenix, Ariz., USA) were used. The injection end of the capillary wasplaced in a liquid filled chamber (15). Light from an argon ion laser(16) (2013-150ML, Uniphase, San Jose, Calif., USA), emitting mainly at488 and 514 nm, was focused by a lens (17) and illuminated the capillaryat 90°. The laser power hitting the capillary was estimated to about 3mW. The polymer coating was not removed at the illumination zone. Theaxial illumination length of the laser in the capillary was estimated to25 μm, and so the detection volume was estimated to 0.2 nl. Light wasguided about 5 cm to the end of the capillary, which was placed in aliquid filled chamber (18). Light exiting the end of the capillary wascollected, end-on, by a condenser lens (19) (063098, Spindler & Hoyer,Göttingen, Germany). Primary light was filtered by one or two low passglass filters (20) (OG 530, Schott Glaswerke, Mainz, Germany). The lightwas collected by a 50 mm camera objective (21) (Series E 1/1.8, Nikon,Tokyo, Japan) onto the surface of a CCD (22) (TE/CCD-1024-TKB/1,Princeton Instruments, Trenton, N.J., USA). In some experiments, a prism(23) (336675, Spindler & Hoyer) was placed in between the glass filterand the camera objective to obtain spectral resolution. The collectedimages were stored and evaluated by means of WinView software (PrincetonInstruments) on an IBM-compatible PC.

Example 1

In this example, the prism (23) was left out of the optical setup, andthe collected light was not spectrally resolved. First, water was pumpedcontinuously through the capillary for a few minutes by means ofpressurised air. Then, a 0.7 nM solution of fluorescein (16630-8,Aldrich, St. Louis, Miss. USA) in water was pumped in the same way. Theexposure time of the camera was 1 s. The recorded trace is shown in FIG.12. The obtained signal was calculated as the difference between thenumber of counts, summed over a number of pixels, for fluorescein andwater, respectively. The noise was calculated as the standard deviationof the water baseline. The concentration detection limit (taken as 3×the noise) was 2.7 pM, and the mass detection limit was 550 ymoles. Theexample shows the efficient collection and detection of fluorescentlight, and demonstrates the excellent detection limit obtained with thedevice.

In FIG. 13, images of the end of a capillary are shown. FIG. 13a showsthe image for pure water. The main feature is a circle of light close tothe circumference of the column, due to light scattering at the outersurface. FIG. 13b shows the image for fluorescein. The main feature isan approximately Gaussian light peak in the center of the column, due toemission of fluorescent light. The peak is superimposed on thering-shaped background. Clearly, by only reading out pixels close to thecenter, it is possible to reject stray light through spatial filtering.

Example 2

In this example, the prism (23) was used. The column was filled with acrosslinked hydrogel (poly(dimethylacrylamide), 7% T, 4% C). The lengthof the capillary from the injection end to the illumination zone wasabout 30 cm. The liquid chambers were filled with a buffer consisting of0.1 M Tris, 0.1 M borate, 2 mM EDTA, and 7 M urea. An 0.084 nM solutionof fluorescein (F-1130, Molecular Probes, Eugene, Oreg., USA) in waterwas electrokinetically injected at 4 kV for 20 seconds, andelectrophoresed at 5 kV. The exposure time was 0.6 s. FIG. 14 shows partof the electropherogram. The concentration detection limit for thefluorescein peak was 70 fM. Again, the example demonstrates theexcellent detectability of the device.

Example 3

The same optical set-up, column, and electrophoresis conditions as inthe previous example were used. The sample was a cycle sequencing DNAsample, precipitated in ethanol and dissolved in water. The primers werelabelled with four different fluorophores (FAM, JOE, TAMRA, ROX)(Genpak, Brighton, UK) and pooled together before the analysis. Theindividual pixels of the CCD-chip were binned into larger superpixels.The spectrum of each data point was recorded as a ten superpixelspectrum in the approximate region 520-670 nm. Wavelength calibrationwas performed by shining light from light emitting diodes of knownwavelength onto the injection end, and recording the obtained spectra.The sequence was evaluated by identifying a number of pure peaks of eachbase from the known sequence, and taking the spectrum for these peaks asrepresentative of the pure bases. Then, the base composition of theremainder of the electropherogram was calculated by mathematical fittingof these calibration spectra to each of the data points. FIG. 15 showsthe so obtained base sequence. The base calling accuracy is estimated to96-98% in the region 40-450 bases. The example shows the excellentdetectability, the negligible detector band broadening contribution, andthe wavelength resolution ability of the device, and clearlydemonstrates the applicability to DNA sequencing.

The invention is, of course, not restricted to the aspects, embodiments,and variants specifically described above, or to the specific examples,but many changes and modifications may be made without departing fromthe general inventive concept as defined in the following claims.

What is claimed is:
 1. A device for detection of one or severalfluorescent species, said species being contained in a medium, saidmedium being contained in a conduit, said device comprising a means ofexciting the fluorescent species by light in an illumination zone, andmeans of collection and detection of fluorescent light emitted by saidfluorescent species, said medium and conduit making up a structure thatis transparent to the exciting light and to fluorescent light emitted bysaid fluorescent species, and said device comprising several suchstructures, characterised in that the refractive index of said medium islarger than that of said conduit or characterised in that said conduitis made of fused silica, quartz, or an organic polymer which conduit hasan external organic polymer coating which has a refractive index lowerthan that of the fused silica, quartz, or organic polymer and whichcoating is transparent to the exciting light, so that at least part ofthe emitted fluorescent light is guided away from the illumination zoneby total internal reflection in said structures and collected from oneend of said structures.
 2. The device according to claim 1, wherein saidconduit is made of fused silica and has a fluoropolymer coating.
 3. Thedevice according to claim 1, wherein the main component of said mediumis water, and that said conduit is made of an organic polymer.
 4. Thedevice according to claim 3, wherein the organic polymer is afluoropolymer or a silicone polymer.
 5. The device according to claim 1,wherein the main component of said medium is an organic liquid, and saidconduit is made of glass, fused silica, or quartz.
 6. The deviceaccording to claim 1, wherein a multitude of said structures arearranged in the form of a planar array, at the illumination zone.
 7. Thedevice according to claim 6, wherein the exciting light is spatiallydispersed across said array by means of one or several lenses, a beamexpander, or a diffractive beam shaper.
 8. The device according to claim6, wherein the exciting light is scanned across said array.
 9. A deviceaccording to claim 6, wherein the light collection ends of said arrayare geometrically rearranged in the form of a two dimensional array. 10.The device according to claim 1, wherein the light that is collectedfrom the end of said structure is detected by an imaging light detector.11. The device according to claim 10, wherein the imaging light detectoris a charge transfer device or a photodiode array.
 12. The deviceaccording to claim 1, wherein the device additionally comprises meansfor spectral resolution of the light that is collected from the end ofsaid structure.
 13. The device according to claim 12, wherein the meansfor spectral resolution Is one or several prisms, gratings, or opticalfilters.
 14. The device according to claim 9, wherein the collectedlight is spectrally resolved by means of one or several optical filters.15. The device according to claim 9, wherein said two dimensional arrayis sparse enough to allow for the collected light to be spectrallyresolved onto the surface of an imaging detector by means of one orseveral prisms or gratings.
 16. The device according to claim 1,additionally comprising a means for spatial resolution of the light thatis collected from the end of said structure.
 17. The device according toclaim 16 wherein the light is spatially resolved by use of an apertureor by rejecting part of a detected image.
 18. The device according toclaim 1, wherein the exciting light is focused in a direction parallelto the guiding direction of the emitted fluorescent light along saidlight guiding structure.
 19. The device according to claim 1, whereinthe exciting light is light from a laser.
 20. The device according toclaim 1, wherein the angle between the propagation direction of theexciting light and the guiding direction of the emitted fluorescentlight along said light guiding structure is orthogonal, to prevent anynon-scattered component of the exciting light to be optically coupledinto the guiding direction of the light guiding structure by totalinternal reflection.
 21. The device according to claim 1, wherein thedistance between the illumination zone and the light collection end ofsaid structure is large enough to allow light rays emanating from theillumination zone, which do not fulfil the conditions for total internalreflection, to be efficiently transmitted out of the light guiding partof said structure before reaching the light collection end.
 22. Thedevice according to claim 21, wherein the distance between theillumination zone and the light collection end of said structure is atleast four times, at least eight times, or at least sixteen times,larger than the largest cross sectional dimension of the light guidingpart of said structure.
 23. The device according to claim 1 wherein saidconduit has the shape of a hollow cylinder.
 24. The device according toclaim 23, wherein the inner diameter of said cylinder is less than orequal to 500 μm, or less than or equal to 100 μm.
 25. Device accordingto claim 1 comprising means for identifying individual structures bycoupling light by total internal reflection into the structures at somepoint other than the light collection end and collecting said light fromthe light collection ends of said structures.
 26. Use of the deviceaccording to claim 1 for detection of species with native fluorescenceor species labelled with one or several fluorophores.
 27. Use of thedevice according to claim 1 for detection in connection with any methodinvolving the transportation of said species across the illuminationzone within said conduit.
 28. Use of the device according to claim 1 fordetection in connection with capillary electrophoresis, includingcapillary zone electrophoresis, capillary gel electrophoresis, micellarelectrokinetic capillary chromatography, and capillary isoelectricfocusing, capillary electrochromatography, liquid chromatography, orflow injection analysis.
 29. Use of the device according to claim 1 fordetection in connection with nucleic acid analysis.
 30. Use of thedevice according to claim 1 for detection in connection with DNAsequencing.